This application builds upon substantial advances in the last period that uncovered a unique chromatin structure of yeast heterochromatin, developed a powerful new assay capable of detecting transient lapses in heterochromatin function, and overturned multiple widely-held views of silenced chromatin. This new assay opened the window to studies of the dynamics of heterochromatin that have previously been elusive. In the upcoming period, five specific aims will address the most important unresolved issues regarding the structure, stability and epigenetic inheritance of silenced chromatin, and reveal what happens to its silencing capacity when it serves as a donor for double-strand break repair. First, the PI and his team will integrate results from their earlier studies of cell- cycle stages in the assembly of silenced chromatin with their high-resolution chromatin mapping and determine which features of the final structure accompany each stage of its assembly and maturation. Second, they will continue a very promising test of whether nucleosomes are carriers of the epigenetic information for the inheritance of silenced chromatin, testing first how the inheritance of silenced chromatin is influenced by the size of the silenced domain. Third, they propose ground-breaking experiments to address the fundamental questions of all chromatin-based epigenetic inheritance by developing broadly applicable technology to map the DNA positions of individual nucleosomes and the proteins that bind them through a round of DNA replication. Forth, they integrate their experience with microfluidics to study at the single-cell level how various proteins functioning at or near the replication fork impact the establishment, maintenance and inheritance of silenced chromatin. Finally, they address the enigma of how silenced chromatin retains its silencing capacity in the face of the molecular invasions accompanying its role as a donor in double-strand break repair. The most proximal health relatedness of this work lies in the growing recognition of the importance of epigenetic processes in cancer. In addition much of development is, by definition, based upon epigenetic processes. Hence the work is highly relevant to many defects in developmental processes.